9/9/2023 0 Comments Ecl solution western blotA recommended first exposure time is 60 seconds. Never place a blot on developed film, as there may be chemicals on the film that will reduce signal.ġ0.Carefully place a piece of film on top of the membrane.Use gloves during the entire film-handling process.Make sure excess substrate is removed from the membrane and the membrane protector.For optimal results, perform the following precautions: Turn off all lights except those appropriate for film exposure (e.g., a red safelight).įilm must remain dry during exposure. Use an absorbent tissue to remove excess liquid and to carefully press out any bubbles from between the blot and surface of the membrane protector.ĩ.Place the protected membrane in a film cassette with the protein side facing up. Typical laboratory lighting will not harm the Working Solution.ħ.Incubate blot with Working Solution for 5 minutes.Ĩ.Remove blot from Working Solution and place it in a plastic membrane protector a plastic sheet protector or plastic wrap may be used. Solution in an amber bottle and avoid prolonged exposure to any intense light. The Working Solution is stable for 8 hours at room temperature.Exposure to the sun or any other intense light can harm the Working Solution. Use 0.1 ml Working Solution per cm2 of membrane. Increasing the wash buffer volume and/or the number of washes may help reduce background.Briefly rinsing membrane in wash buffer before incubation will increase wash efficiency.Ĥ.Incubate blot with the appropriate HRP-conjugate dilution for 1 hour at RT with shaking.ĥ.Repeat Step 3 to remove non-bound HRP-conjugate.Membrane must be thoroughly washed after incubation with the HRP-conjugate.Ħ.Prepare Working Solution by mixing equal parts of the Stable Peroxide Solution and the Luminol/Enhancer Solution. ![]() Wash membrane by suspending it in Wash Buffer and agitating for 5 minutes. Incubate blot for 1 hour with shaking.If desired, blots may be incubated with primary antibody overnight at 2-8☌.ģ. Remove the Blocking Reagent and add the appropriate primary antibody dilution. For best results, block for 1 hour at RT.Ģ. Remove blot from the transfer apparatus and block nonspecific sites with Blocking Reagent for 20-60 minutes at room temperature (RT) with shaking.
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